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microRNA-1 (miR-1) is a well-studied conservative microRNA (miRNA) involved in immune responses in mammals and insects. However, little is known about its role in pesticide resistance in arthropods. In this study, we found that a microRNA belong to miR-1 family (tci-miR-1-3p) was significantly down-regulated in a cyflumetofen-resistant strain (CYR) of Tetranychus cinnabarinus compared with its homologous susceptible strain (SS), indicating an involvement of miR-1 in cyflumetofen resistance in mites. One glutathione S-transferase (GST) gene (TCGSTM4, a mu class GST gene), a candidate target gene of tci-miR-1-3p, was found to be significantly down-regulated when tci-miR-1-3p was over-expressed. The specific interaction between tci-miR-1-3p and the target sequence in the 3' untranslated region of TCGSTM4 was confirmed. A decrease or increase in tci-miR-1-3p abundance through feeding miRNA inhibitors or mimics significantly increased or decreased TCGSTM4 expressions at the mRNA and protein levels, respectively. In addition, an over-expression of tci-miR-1-3p resulted in a decrease in the tolerance of T. cinnabarinus to cyflumetofen in both SS and CYR strains, and vice versa. After decreasing TCGSTM4 transcription via RNA interference, T. cinnabarinus became more sensitive to cyflumetofen in both resistant and susceptible mites, and the change in mortality was greater in CYR than that in SS. Moreover, the recombinant TCGSTM4 could significantly decompose cyflumetofen, indicating that TCGSTM4 is a functional gene responsible for cyflumetofen resistance in mites.

BACKGROUND Cyflumetofen is an outstanding acaricide with a novel mode of action. Tetranychus cinnabarinus, an important agricultural pest, is notorious for developing resistance to most classes of acaricides rapidly and results in enormous loss for the economy. Our previous study had pointed out glutathione S‐transferase (GSTs) significantly contributed to the cyflumetofen‐resistance formation in T. cinnabarinus, but the more specific mechanism needed to be further investigated. RESULTS The unstable resistance was observed in cyflumetofen‐resistant strain (CyR)under acaricide‐free condition. The activity of GSTs increased along with the development of resistance. The expressions of 13 GST genes were detected in CyR and susceptible strain (SS), of which six genes were overexpressed in CyR and the TcGSTm02 was selected as the representative for functional study. The expression of TcGSTm02 changed along with the resistant level of CyR with the same trend. Recombinant protein of TcGSTm02 with high activity was successfully obtained by E. coli expression system, whose activity could be inhibited by cyflumetofen (IC50 = 0.23 mM). Recombinant TcGSTm02 could effectively decompose cyflumetofen, and catalyze GS− to conjugate with cyflumetofen. CONCLUSION All clues confirmed that GSTs strongly associated with cyflumetofen‐resistance and a representative gene, TcGSTm02, showed function on contributing the evolution of cyflumetofen‐resistance in T. cinnabarinus. © 2019 Society of Chemical Industry

BACKGROUND: Acaricide resistance is a serious problem in spider mites. Cyflumetofen is a new complex II inhibitor, whereas pyridaben acts at complex I and has been used for decades. Although cross-resistance between cyflumetofen and pyridaben has been observed in Tetranychus cinnabarinus, the specific mechanisms at play have not yet been investigated. -resistant strain. In addition, recombinant CYP389C16 (40 pmol) effectively metabolized 25.0 ± 0.7% of cyflumetofen, 39.7 ± 1.0% of pyridaben, and 69.3 ± 3.3% of AB-1 (active de-esterified metabolite of cyflumetofen) within 2 h. In addition, hydroxylation metabolite of AB-1 was identified by HPLC-MS/MS. RESULTS: Investigation into the cross-resistance mechanisms identified five P450s, among which CYP389C16 was evaluated as the most likely candidate conferring cross-resistance. Knockdown of CYP389C16 expression via RNA interference diminished the level of cross-resistance in the cyflumetofen CONCLUSIONS:The study reveals that overexpressed CYP389C16 is involved in the cross-resistance between cyflumetofen and pyridaben in T. cinnabarinus.

BACKGROUND: Increased expression or point mutations of carboxyl/cholinesterases (CCEs) have been involved in many cases of insecticide and acaricide resistance. However, it has been only rarely documented that downregulation of CCE genes is associated with resistance, although many insecticides and acaricides need hydrolytic activation in vivo. Previously, expression analysis of a laboratory-selected cyflumetofen-resistant strain of Tetranychus cinnabarinus indicated that resistance was associated with increased expression of a CCE gene of TcCCE04, but also the downregulation of two CCE genes, TcCCE12 and TcCCE23.RESULTS: Synergism experiments revealed the importance of ester hydrolysis in cyflumetofen toxicity, because treatment with S,S,S-tributylphosphorotrithioate (DEF) caused strong inhibition of cyflumetofen hydrolysis, in both the susceptible and resistant strains. Moreover, silencing expression of TcCCE12 and TcCCE23 via RNAi further decreased the susceptibility of mites to cyflumetofen significantly, suggesting that downregulated CCE genes could be involved in cyflumetofen resistance. In addition, it was shown that recombinant TcCCE12 protein could hydrolyze cyflumetofen effectively. CONCLUSION: Decreased esterase activity via downregulation of specific CCE genes most likely contributes to cyflumetofen resistance by decreased activation of cyflumetofen to its active metabolite. Mixtures of cyflumetofen and esterase-inhibition acaricides (e.g. organophosphates or carbamates) should be avoided in field applications.

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